Hazard identification on a single cell level using a laser beam.

This research shows a novel method for hazard identification of a chemical and UV light on a single cell level with a laser probe beam. The laser probe beam was passed through interface of cell membrane/culture medium of a cultured human hepatoblastoma cell line HepG2. Deflection of the laser probe beam, which was induced by changes in concentration gradients due to the active materials movement across the cell membrane, was monitored. When a toxic hazard existed, a living cell was expected to be killed or injured, or cellular behaviors to be changed greatly. Then, the changing deflection signal from the living cell would become unchanged or altered in a different way. This was successfully demonstrated with cytotoxicity of UV light and H(2)O(2). Most of the cultured HepG2 cells showed changing deflection signals after 10 min illumination of UV-visible light longer than 370 nm, while almost all HepG2 cells showed unchanged deflection signal after 10 min illumination of UV-visible light with wavelength longer than 330 nm. The results suggested that UV light between 330-370 nm could kill the cells. Additions of H(2)O(2) solution with different concentrations to the cell cultures caused the changing deflection signal from a living cell either unchanged or changed in different trend, suggesting toxicity of H(2)O(2) to the cells. The results from the beam deflection detection agreed well with those obtained by the conventional trypane blue method.


Introduction
There are huge numbers of synthesized chemicals, and the potential adverse health effects of human exposures to most of them have not been understood. Identifi cation of whether a chemical is toxic hazard is usually done by human case reports, experimental laboratory animal studies, environmental research, and in vitro studies with cell lines. [1][2][3] Among these methods, in vitro studies with cell lines are the fastest and easiest methods, where cell death or cell injuries or changes in cellular behaviour are examined in presence of a target chemical. [1][2][3][4][5][6][7] The most conventional cell death or cell viability is determined by labelling method using a dye such as trypane blue 8 or fl uorescent dyes. 9 Flow cytometry 10 can provide living or dead information about individual cells, but usually requires fl uorescent staining and cannot be used for direct monitoring of cells in the culture process. Various microscopic techniques such as fl uorescence microscopy, which also requires labelling, have been widely used for observing cells. However, the microscopic techniques are labour-intensive tasks, prone to error and operator fatigue. Vibrational spectroscopies [11][12][13][14] such as the Infrared and Raman spectroscopy are nondestructive and noninvasive methods for study of cells. However, infrared measurements cannot be carried out in cell cultures because of the strong absorption of water in the infrared spectral region. [11][12] Raman signal from biological molecules is usually low and thus the application in cell study is limited. [13][14] Very recently, we have applied the optical beam defl ection (OBD) [15][16][17][18] method to non-invasive diagnosing of a single living cell from a dead one. 19 In this method, a laser probe beam is passed though an area of cell membrane of the cell as shown in Figure 1, and the defl ection of the laser probe beam is monitored. When the cell is alive, movement of active materials across the cell membrane occur. For example, nutrients and oxygen move into, while metabolic products and CO 2 move out of the cell, as shown in left of Figure 1-B. The movement of the materials induce changes in concentration gradients close to the cell membrane. The changes in the concentration gradient generate a change of the refractive index gradient of the medium near the cell membrane, which in turn generate a defl ection of the Correspondence: Xing-Zheng Wu, Department of Materials Science and Engineering, School of Engineering, University of Fukui, Bunkyo 3-9-1, Fukui-shi 910-8507, Japan. Tel: +81-776-27-8610; Fax: +81-776-27-8767; Email: xingzheng.wu@matse.fukui-u.ac.jp laser probe beam. However, for a dead cell, little or no active material movements across the cell membrane occurs. Accordingly, little or no changes in defl ection of the probe beam are generated as shown in right of Figure 1-B. In addition to the concentration gradients, a temperature gradient induced by heat from a chemical or biochemical reaction in cell activities also generates a deflection of the probe beam. However, the defl ection caused by the temperature gradient is usually negligible compared to that induced by the concentration gradient, except for some special cases where a remarkable temperature difference is produced during the cell activities. 19 The OBD diagnosis method for a single cell is based on monitoring of changes in concentration gradients in the vicinity of the cell membrane. In principle, this method can be used for determining any change in active movement of materials across the cell membrane of a single living cell. If a chemical is a toxic hazard to the living cell, the cell is expected to be killed or damaged; or cell activities are greatly affected by existence of the chemical. Then, the active movement of materials across the cell membrane is expected to cease or change greatly. This should be detected by monitoring of the defl ection signal of the probe beam. In this work, we investigate the possibility of applying the OBD technique as a novel tool for fast toxic hazard identification of chemicals. Human hepatoblastoma cell line HepG2 was used as a model cell line. Cytotoxity of UV light and H 2 O 2 on the cultured cells are investigated.

The Experiment
Experimental set-up for the beam defl ection was similar as that reported before. 19 As shown in  bottom was placed on a platform of a microscope (CK40, Olympus of Japan). A laser beam from a diode laser (output power: 3 mW; wavelength: 667 nm) was introduced into the microscope. The probe beam was refl ected inside the microscope, and then focused (spot size, about 3 ∼ 5 µm) to the cell membrane/culture medium interface of a cultured liver cell (about 15 µm in diameter) by an objective lens (N.A.: 0.1). An X-Y stage was used to adjust the distance between the laser beam and the cell membrane. The distance between the laser beam centre and the cell membrane was approximately 2 ∼ 3 µm. The probe beam exited from the culture dish and was focused to the centre of a bi-cell photodiode with a lens (focal length: 10 cm). The distance between the lens and the bottom of the dish was about 15 cm. The photo-currents i 1 and i 2 from the two sides of the bi-cell photodiode were inserted into an electric calculating circuit, where (i 1 -i 2 )/(i 1 + i 2 ) was calculated and fi nally converted into electric voltage (volt) as outputs of the defl ection signal.
The human hepatoblastoma cell line HepG2 was used throughout this work. This cell line expresses a variety of liver functions including albumin production. The medium used was Dulbecco's Modifi ed Eagle (DME) medium (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS), 0.2% sodium bicarbonate, 10 mM N-2-Hydroxyethylpiperazine-N'-2-Ethane Sulfonic Acid (HEPES), 2 mM glutamine, and 0.06 mg ml -1 kanamycin. The cells were grown and adhered on the bottoms of culture dishes at 37 °C in humidifi ed air containing CO 2 at 5%. 20 The viable and nonviable cell densities were conventionally determined by the trypan blue exclusion method using a Neubauer improved hemocytometer (Erma, Tokyo, Japan). Trypan blue was purchased from Dojindo (Kumamoto, Japan).
In experiments of light illumination, light from a high-pressure mercury lamp (Ushio Corp., Japan) was fi rstly passed through a water fi lter of 10 cm, then a cut-off fi lter with cut-off wavelength of 330, 350, or 370 nm, and fi nally illuminated to the culture containing 0.45 ml DME medium. The water fi lter was used for absorption of infrared light to avoid the temperature rise in the cell culture. The cut-off fi lter with cut-off wavelength of 330, 350, or 370 nm, cut off UV light with wavelength shorter than 330, 350, or 370 nm, i.e. it only permitted UV-visible light with wavelength longer than 330, 350, or 370 nm to illuminate the cells. The illumination period was 10 min. After the illumination, the culture dish was placed on the platform of the microscope, and its position was adjusted to let the laser probe beam pass through the interface of the cell membrane/culture medium of a cell. The defl ection signal from the cell was monitored for about 500 s. Then, the position of the dish was readjusted for monitoring the deflection from another cell for 500 s too. This monitoring was sequentially repeated for 10 different cells. At last, cell viability of the illuminated cells in the culture dish was determined by the conventional trypan blue exclusion method.
In experiments of H 2 O 2 hazard identifi cation, the deflection signal from a cell in a culture dish containing 0.45 ml DME medium was fi rstly monitored for about 10 min. Then, 50 µl solution of Η 2 O 2 with certain concentration was added into the DME medium in the culture dish, and the defl ection signal was continually monitored for another 10 min.

Results and Discussions
Because it is known that UV light could induce apoptosis relate to the cell cycle, 21-23 the effect of the UV-visible light illumination to the cultured cells is fi rstly investigated here. Figure 2-A shows results of 10 sequential measurements of defl ection signals from 10 different cells without light illumination, and Figures. 2-B, -C, and -D show those after 10 min illumination of different UV-visible light, respectively. The cells in Figures. 2-A, -B, -C, and -D are cultured at the same time and at the same conditions. Thus they have same cell viability before light illumination. In Figure. 2-A, defl ection signal of sequential No. 3 changed little with time during the monitoring process. This suggested that the cell in sequential No. 3 was a dead one. Defl ection signals in other sequential numbers changed with time although the change in sequential No. 7 and 8 were small. This suggested that they were alive. 19 The different cells showed different deflection signals. This was because that the different cells had different active material movements, and the active material movements always changed with time.
In Figure 2-B, defl ection signals in sequential No. 7 and 8 changed little with time, suggesting cells of the sequential No. 7 and 8 were dead. Others showed the changing defl ection signal with time. This implies that most of the living cells were still alive after 10 min illumination of the UV-visible light with wavelength longer than 370 nm. On the other hand, less than half of the cells showed defl ection signals changing with time in Figure 2-C (the sequential number was not indicated because the plots in Figure 2-C were too close or overlapped each other). This suggested that less than half of the cells were kept alive after 10 min illumination of UV-visible light with wavelength longer than 350 nm. Moreover, changes of the defl ection signals with time in Figure 2-C were much smaller than those in Figures 2-A and 2-B, suggesting that the active materials movements of the living cells had changed greatly due to the light illumination with wavelength longer than 350 nm. In Figure 2-D, most of cells showed that the defl ection signals changed little with time (the sequential number was not indicated too because of the overlap of the plots), suggesting that most of the them had died after the illumination of UV-visible light with wavelength longer than 330 nm. The results of Figure 2 suggest that UV light between 330 ∼ 370 nm has a strong killing effect on the liver cells.
The traditional trypane blue method was also used for determinations of the cell viabilities with and without the light illuminations. Table 1 shows the cell viabilities obtained by the trypan blue method.
The cell viabilities did not change after the illumination of the UV-visible light with wavelength longer than 370 nm. This indicates that 10 min illumination of UV-visible light long than 370 nm would not kill the cells. On the other hand, the cell viabilities were decreased to 22 and 6% when the UV-visible light wavelengths were longer than 350 and 330 nm, respectively. This means that the 10 min illumination of the UV-visible light with wavelength longer than 350 and 330 nm could kill about 71 and 87% living cells, respectively, in comparison with the results without or with light illumination with wavelength longer than 370 nm. These results indicate that the after illuminated with light >370 nm  UV light between 330 ∼ 370 nm has strong toxic effect to the living cells, and concurred with the conclusions obtained by the OBD method. The method also can be used to real time monitoring of the UV illumination effect on a single living cell. The defl ection detection system is constructed by introducing an UV illumination source directly into the microscope and the results will be reported later.
The method is further applied to assess toxicity of chemicals to the living liver cells. Hydrogen peroxide was chosen as a model chemical because it can induce apoptotic-like cell death, 24 oxidative cell injury, 25 and killing of cultured hepatocytes. 26 Figure 3 shows changes of defl ection signals of living cells before and after additions of different concentrations of H 2 O 2 to cell cultures. In Figures  3-A, B, C, all of the defl ection signals were changing with time before the addition of H 2 O 2 . This meant that the cells were alive before the addition of H 2 O 2 . However, after the addition of H 2 O 2 , the defl ection signals were basically on the level of the dash lines as shown in Figures 3-A and 3-B although there are some noises. That is, the changing defl ection signal remained unchanged. This implied that the living cells died soon after the concentration of H 2 O 2 in the cell cultures increased from 0 to 0.03 and 0.003%. On the other hand, the defl ection signal continued to change with time when the concentration of H 2 O 2 was 0.0003% in Figure 3-C, but with different changing trend, compared with the results before the addition of H 2 O 2 . This suggested that although the cell was still alive, but it might have greatly damaged or injured after the concentration of H 2 O 2 in the culture medium increased from 0 to 0.0003%. Figure 3 suggests that H 2 O 2 is strongly toxic to the cultured cells and its toxicity increased with its concentration.  The living cell densities cultured in different concentrations of H 2 O 2 were also determined by the trypane blue method. Table 2 shows that living cell densities decreased with the H 2 O 2 concentrations. When the H 2 O 2 concentration in the culture medium increased from 0 to 0.0003, 0.003, and 0.03%, the living cell density decreased from 2.27 to 1.46, 0.67, and 0.022 × 10 4 cells/well, respectively. This also suggests that H 2 O 2 is a toxic hazard to the cells, and its toxicity increased with its concentration.
Cell culture conditions are expected to affect the cell densities and cell activities greatly. Effects of the H 2 O 2 and UV-visible light on beam defl ection from living cells and the cell viabilities cultured under different culture conditions (for example, different CO 2 concentrations rather than 5% used in this work) will be investigated in near future and results will be reported later.
As conclusion reported above, the conventional trypane blue method gave changes in cell viabilities or densities before and after treatment of a chemical or UV light illumination. Information on how many living cells had been killed by the treatment is able to be determined, however dynamic information related to the toxic effect of the chemical has not been established. The conventional method cannot be used for real-time monitoring of the toxic effect of a chemical on a single cell level. On the other hand, the OBD method is based on the real-time monitoring of change in concentration gradients near the cell membrane of a single living cell. The monitored OBD signal basically refl ects dynamic change on the active material movements across the cell membrane before and after the treatment of the chemical. In addition to the real-time monitoring, cell viabilities also can be obtained by repeating the monitoring of the defl ection signals from different cells, as reported in our previous paper. 19 This method is expected to be particularly useful in probing changes of the material movements across the cell membrane in dying or damage processes induced by chemicals. 0 9 × 10 -5 3 × 10 -4 9 × 10 -4 3 × 10 -3 9 × 10 -3 3 × 10 -2 9 × 10 -2 Living cell density ( × 10 4 cells/well) 2.27 1.86 1.46 1.32 0.67 0.26 0.022 0